A multiplex qPCR assay for the identification of Pinus palustris and Pinus elliottii
Abstract
Differentiating the wood of high-strength southern yellow pines (Pinus palustris and P. elliottii) from their lower-strength congeners (P. taeda and P. echinata) presents a substantial challenge once diagnostic external features, such as needles, cones, and bark are absent. This difficulty arises from their wood anatomical indistinguishability and low genetic resolution driven by highly conserved genome organization and extensive allele sharing. To address this, we developed and validated a multiplex qPCR assay utilizing SYBR Green chemistry and asymmetric primer concentrations, working with leaf and seed DNA. The assay targets short amplicons (<100 bp) to maximize the likelihood of amplification of the fragmented DNA typical of solid wood. The method achieved high linearity (R² > 0.99) and efficiency, successfully co-amplifying targets while maintaining clear discrimination through distinct mean melting temperatures (77.98°C [SD 0.11] and 69.25°C [SD 0.26], respectively). Sensitivity testing established limits of detection at 1 pg for Pinus palustris and 10 pg for Pinus elliottii. Validation across a panel of 84 individuals representing all four species yielded 100% accuracy, precision, and recall. This study establishes a rapid, gel-free molecular diagnostic tool that overcomes the limitations of wood anatomical identification. By validating a short-amplicon design on leaf and seed tissues, it provides a proof-of-concept for future applications on solid wood and offers a reproducible methodological framework for species verification.
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